Abstract

Muscle injury or modified muscle use can stimulate muscle invasion by leucocytes that have the potential to increase tissue damage or promote tissue growth and repair. In the present investigation, we examined the role of macrophages in muscle injury, repair and regeneration during modified muscle loading. Weight-bearing was removed from the hindlimbs of mice for 10 days followed by reloading through normal ambulation. During the unloading period, soleus muscle fibre cross-section decreased by 38%. Prior to the onset of reloading, mice received a series of intraperitoneal injections of anti-F4/80, which binds a mouse macrophage surface antigen. Although anti-F4/80 injections did not affect macrophage numbers in soleus muscles at 2 days of reloading, macrophages were reduced by 86% at 4 days of reloading. Muscle membrane lysis during the reloading period did not differ at 2 days of reloading between anti-F4/80-treated mice and mice that received isotype control antibody. However, control animals showed large decreases in the number of fibres with membrane lesions at 4 days of reloading, but this membrane repair did not occur in macrophage-depleted mice. Macrophage-depletion also reduced muscle regeneration (indicated by central nucleation) and satellite cell differentiation (indicated by reductions in MyoD-expressing satellite cells) and prevented growth of muscle fibres that normally occurred in control animals between days 2 and 4 of reloading. These findings collectively show that macrophages play a significant role in muscle fibre membrane repair, regeneration and growth during increased muscle use after a period of atrophy.

Macrophage concentrations in soleus muscles of mice subjected to 10 days of hindlimb muscle unloading, followed by muscle reloading during normal ambulation

Bars show s.e.m. *Significantly different from the isotype IgG control group during the same period of reloading; #significantly different from the 2 day reloaded group that received the same experimental treatment at P < 0.05.

Macrophage depletion increases muscle fibre membrane lesions in the soleus muscles of mice subjected to 10 days of hindlimb unloading, followed by 4 days of muscle reloading

A, data are the percentage of total fibres in the cross-sections of entire soleus muscles that were brightly fluorescent following procion orange treatment. Bars show s.e.m. *Significantly different from the anti-F4/80-injected group at the same stage of reloading; #significantly different from the 2-day reloaded group that received the same antibody treatment. B, the peaks show the aggregate data for measurements of intracellular fluorescence for all fibres in cross-sections of the entire soleus muscle from all mice in each treatment group. A rightward shift of peaks on the abscissa indicates an increase in fibres with membrane lesions. Note that the background fluorescence set at intensity = 0 was determined by measuring fluorescence at a region of the microscope slide where there was no tissue. Thus, even non-injured, ambulatory fibres displayed a small positive value for fluorescence. The dark peak shows data from anti-F4/80-treated mice after 4 days of reloading. The light peak shows data from isotype control antibody-treated mice after 4 days of reloading. Solid line shows mean fluorescence intensity of fibres from ambulatory animals. Dashed line shows mean fluorescence of fibres from isotype control IgG- and anti-F4/80-treated fibres after 2 days reloading, which did not differ between the two groups.

Western blot showing that dysferlin expression remained elevated after 4 days of reloading in the soleus muscles of mice that were macrophage depleted

Each lane was loaded with 30 μg total protein. Row A, anti-dysferlin blot of muscle extracts from non-depleted mice. Row B, anti-dysferlin blot of muscle extracts from macrophage depleted mice. Row C, same blot as shown in row A, but reprobed with anti-desmin to show relative quantities of desmin in each lane, as a loading control. Row D, same blot as shown in row B, but reprobed with anti-desmin to show relative quantities of desmin in each lane, as a loading control. AMB, ambulatory controls; HS, hindlimb suspended only; 2 day, 2 day reloaded; 4 day, 4 day reloaded.

The increase in regenerative fibres in soleus muscles that occurs between 2 and 4 days of reloading does not occur in macrophage-depleted mice

The percentage of total muscle fibres in muscle cross-sections that were centrally nucleated was used as an index of regeneration. *Significantly different from control at P < 0.05; #significantly different from treatment-matched 2 day sample at P < 0.05.

A, the number of satellite cells in macrophage-depleted soleus muscle is significantly greater than in non-depleted muscle at 2 days of reloading. B, MyoD-positive satellite cells decrease during the interval between 2 and 4 days of reloading in non-depleted but not in macrophage-depleted soleus muscles. *Significantly different from control at P < 0.05; #significantly different from treatment-matched 2 day sample at P < 0.05.

Muscle fibres in the soleus muscle from mice treated with isotype control antibody and from mice treated with anti-F4/80

Muscle fibres in the soleus muscle of mice treated with isotype control antibody increased in cross-sectional area during the interval between 0 and 4 days of reloading. However, fibres in muscles from mice treated with anti-F4/80 do not grow in cross-section during the interval between 2 and 4 days of reloading. *Significantly different from treatment-matched sample in 0 h reload group at P < 0.05; #significantly different from treatment-matched 2 day sample at P < 0.05.